Human CLL Intraclonal Fractions Differ in Their Abilities to Respond to, Elicit, and Suppress Pro-Engraftment and Growth Signals From Autologous T Cells in a Murine Adoptive Transfer Model

Abstract 316 Chronic lymphocytic leukemia (CLL) clones contain activated/proliferative leukemic cells in lymphoid tissues and resting cells in the periphery. Different subsets of CLL cells have distinct proliferation rates. Recently divided “proliferative” cells have a surface membrane phenotype of CXCR4 DIM CD5 BRIGHT (CXCR4 DIM ) and contain higher numbers of CD38 + and Ki-67 + cells. Circulating “resting” CLL cells express CXCR4 BRIGHT CD5 DIM (CXCR4 BR ) and genetic signatures of older, quiescent cells that need to home to lymphoid tissues or die. CXCR4 DIM and CXR4 BR subsets are relatively minor (1–10% of total) components of CLL clones, with the major fraction (≥90%) of CLL cells having intermediate levels of CXCR4 and CD5 (CXCR4 INT ). Based on these differences, we proposed a model of transitioning CXCR4 DIM → CXCR4 INT → CXCR4 BR CLL cells in the blood. Because higher birth rates correlate with more aggressive disease, and transiting back to solid tissues permits clonal survival and re-activation, this model suggests CXCR4 DIM and CXCR4 BR subsets as therapeutic targets. Aiming to further understand functional differences in CLL subsets in vitro and in vivo , we found that CLL subsets differ in cell size (CXCR4 DIM >CXCR4 INT >CXCR4 BR ), in vivo apoptosis and transmigration in vitro (both CXCR4 DIM INT BR ). Thus, while more CXCR4 BR cells undergo apoptosis, CXCR4 BR cells can migrate better to tissues to receive survival signals. In vivo functional differences were then studied in a NOD/SCID/γc null (NSG) mouse model using pre-activated CLL-derived autologous T cells. Primary CLL blood cells from 1 M-CLL and 2 U-CLL patients were sorted for CXCR4 BR , CXCR4 INT or CXCR4 DIM fractions. Each fraction (5×10 6 cells) was injected into NSG mice with 5×10 5 CD3/28-activated autologous T cells. At weeks 2–6 post transfer, blood analyses showed more extensive expansion of CLL B and T cells in mice received CXCR4 DIM than in those injected with CXCR4 BR or CXCR4 INT . At weeks 9–12, mice were sacrificed. Although T cells dominated in blood, spleen and bone marrow of all recipients, a larger fraction of CLL B cells existed in CXCR4 BR injected mice, suggesting better long-term CLL cell engraftment capacity of this fraction. Because regulation of T cells plays key roles in CLL cell survival/growth in patients and in the NSG adoptive transfer model, we next analyzed the same fractions for their abilities to activate T cells and elicit help for engraftment and growth. Unactivated CD5 + T cells (1–1.5×10 5 ) and B-CLL fractions (3–5×10 6 cells) were sorted from 6 patient samples (3 U-CLL and 3 M-CLL), injected into mice and followed bi-weekly until week 6. In 5 cases, except one with few CXCR4 BR and CXCR4 DIM cells, CXCR4 DIM injected mice had more extensive T cell growth starting from week 2. Mice injected with CXCR4 BR from 2 U-CLL cases also showed T cell expansions, but at comparatively lesser levels and at later time points (from week 4–5). At week 6, CLL B cells were found in spleen and bone marrow in mice with activated T cells; the numbers of CLL B cells correlated with T cell numbers. Also, identical CXCR4 levels were found in CLL cells regardless of origination from CXCR4 BR or CXCR4 DIM . Notably, no human B or T cells were detected in CXCR4 INT injected mice. In fact, adding CXCR4 INT cells to CXCR4 DIM mice suppressed CXCR4 DIM induced T cell expansion and cytokine production. Specifically, mice receiving both CXCR4 DIM and CXCR4 INT cells had diminished T cell expansion and at least 3 fold reduced serum levels of IFNγ and IL5. Overall, our data confirm the need for activated T cells for CLL B cell growth in mice; suggest superior long term CLL B cell engraftment by CXCR4 BR cells with activated T cell support, and identify a greater ability of CXCR4 DIM cells to activate autologous T cells, although some U-CLL CXCR4 BR cells could do so to a lesser degree. Superior activation of T cells by CXCR4 DIM B cells may be due to higher numbers of CD23 + , CD25 + , CD27 + , CD29 + and CD44 + cells in CXCR4 DIM fraction that facilitate cellular interactions. Finally, unlike CXCR4 BR and CXCR4 DIM cells, the major fraction in patient blood, CXCR4 INT , inhibited T cell activation. These results indicate previously unappreciated levels of intraclonal CLL cell heterogeneity that may have important clinical relevance, allow more precise biologic analyses, and provide a rationale for preferential therapeutic targeting of these fractions. Disclosures: No relevant conflicts of interest to declare.